PROJECT SUMMARY/ABSTRACT This research will utilize spherical nucleic acid (SNA) nanostructures to develop effective, structure-informed vaccines for advanced melanoma. Traditional treatments (i.e. chemotherapy or radiation) are less successful for melanoma because of difficulties in discerning melanoma cells phenotypically. Immunotherapeutics must ensure that melanoma—with a high mutational burden—cannot easily evade the immune system. SNAs can function as robust cancer vaccines through the precise control over the presentation of multiple melanoma-associated targets to immune cells which lowers its potential for immune evasion. SNAs are composed of a nanoparticle core with a dense radial shell of nucleic acids. When synthesized using immunostimulatory “adjuvant” oligonucleotides, SNAs induce immune responses. Indeed, this adjuvant only structure demonstrates the enhanced responses generated from a 3D structure compared to linear adjuvant and forms the basis of an ongoing Phase 1b/2 clinical trial. We have exploited the ease of chemical synthesis and modular architecture of SNAs, and synthesized them to include both adjuvant and a single tumor-associated peptide (“antigen”). These structures enhance antitumor responses and provide long-term protective immunity in model systems, and in particular, there is a strong relationship between vaccine structure and efficacy. In our proposed work, we aim to develop SNA vaccines against melanoma by precisely incorporating and presenting multiple immunostimulatory cues to the immune system. SNAs will be synthesized with multiple clinically-relevant melanoma antigens (MHC-I and -II restricted, tumor-associated, neoantigens), with structural variations in how the adjuvant and antigen are presented. Control over structure, combined with in vitro and in vivo evaluations of immunostimulation, will elucidate structure-activity relationships that will inform the future of cancer vaccine design. Using structure to control the presentation of multiple immune system cues has the power to elevate immune responses to melanoma and improve clinical outcomes. In Aim 1, we will synthesize SNAs containing multiple antigens and varied stabilities to enhance antigen-specific T cell responses. We will analyze their uptake by immune cells, subcellular trafficking of the SNA components, and kinetics of activation of multiple pathways (e.g. antigen presentation, co-stimulatory marker expression). In Aim 2, we will compare different administration routes and analyze in vivo biodistribution and uptake kinetics, as well as the antigen-specific immune responses raised by SNAs in immunocompetent mice. We will assess raised responses after delivery of SNAs containing human antigens to humanized mice and patient specimens. In Aim 3, we will evaluate SNA antitumor efficacy in vivo alone and in combination with immune checkpoint blockade, and identify SNAs as candidates for further preclinical studies and clinical translation. Significantly, this approach will generate a structure-based understanding of SNA performance as vaccines, and improve immunotherapy by generating a breadth of T cell responses with superior efficacies.

PROJECT NARRATIVE The proposed research will develop a new, promising, and clinically relavant class of nanotherapeutics – spherical nucleic acids (SNAs) – as immunostimulatory agents for treating advanced melanoma, a disease where traditional cancer treatments including chemotherapy and radiation are largely ineffective, thereby significantly and positively impacting human health. SNAs offer an innovative approach to elucidate nanoscale structure-activity relationships, and deliver multiple immunostimulatory cues for generating enhanced antitumor responses. Broadly, this work will inform the rational design of new classes of cancer vaccines, resulting in significant advances in immunotherapy development for various forms of cancers.